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High level of activated or phosphorylated serine/threonine kinase Akt is commonly observed in aggressive breast cancer. Hence, the Akt signaling pathway has become a popular target for therapeutic interventions. Previously our lab discovered that phosphorylated Akt is co-expressed with a transcription/translation factor called the Y-box Binding Protein-1 (YB-1) and that Akt phosphorylates YB-1 at Ser102 in vitro. It was also found that YB-1, but not the YB-1 mutant in which Ser102 is mutated to alanine (YB-1A102), enhanced breast cancer cell growth. Since YB-1 was originally isolated as a DNA binding protein that interacts with the regulatory elements of the epidermal growth factor receptor (EGFR), we addressed the possibility that phosphorylated YB-1 stimulates breast cancer cell growth by up-regulating EGFR expression. First, we demonstrated that YB-1 was phosphorylated through the PI3K/Akt pathway ex vivo by immunoprecipitation and western blotting. We then illustrated that loss of Ser102 affects the nuclear translocation of YB-1, implying that Akt may also regulate YB-1 nuclear trafficking. By exogenously expressing YB-1 or YB-1A102 in breast cancer cell lines we showed that YB-1 but not YB-1A102 induced the levels of EGFR mRNA and protein. It was revealed that YB-1 bound to the -2kb of the EGFR promoter by chromatin immunoprecipitation. Interestingly, disruption of Ser102 prevented YB-1 from interacting with the first 1 kb of the EGFR promoter, indicating that binding to this region by YB-1 is regulated by the PI3K/Akt signaling. We then demonstrated that mutation of Ser 102 perturbed YB-1 from activating the -1kb of the EGFR promoter by luciferase reporter assays. Finally, since activation of Akt depends on 3-phosphoinositide-dependent protein kinase-1 (PDK1), we examined the potential of a PDK1 inhibitor, OSU-03012, to inhibit the functions of YB-1. It was found that OSU-03012 blocked YB-1 nuclear translocation and binding to the -1 kb promoter of EGFR. Together these results suggested that activation of the PI3K/Akt pathway leads to phosphorylation of YB-1 at Ser102, resulting in up-regulation of EGFR gene expression. Here we propose that Akt, YB-1, and EGFR may all function in the same pathway to promote breast cancer cell growth.